Technical Notes Anal. Chem. 1995, 67,2279-2283
Artifacts Related to Sample Introduction in Capillary Gel Electrophoresis Affecting Separation Performance and Quantitation Andrds GUttman*st and Hetbrt E. Schwartz* Beckman Instruments, Inc., 2500 Harbor Boulevad, Fullerton, Califomia 92634,and Palomar Analytical Services, 150 Montalvo Road, Redwood City, Califomia 94062
Two injection-related artifacts with capillary gel electrophoresis (CGE) are reported. The first occurs with consecutive injections from the same, low-volume (10200 pL), aqueous sample: progressively smaller peak heights are obtained with each injection. This phenomenon is explained in qualitative terms by examining the electrochemical processes occurring during the electrokinetic sample introduction. A simple solution to this problem is to perform an intermediate electrokinetic injection from a water vial prior to the sample injection. This two-step injection procedure results not only in dramatically increased precision (important for quantitative studies) but also in increased sample loading. The second artifact observed in CGE is related to the physical shape of the inlet of the gel-filledcapillary: employhg an oblique edge capillary results in poor performance, peak distortion, and loss in resolution. In capillary gel electrophoresis (CGE), two types of capillary columns are presently used.’” Those of the first type, involving relatively low-viscosity polymer networks such as those consisting of linear polyacrylamide or alkylcellulose,are often referred to as “replaceable”,as the gel can be rinsed in and out of the capillary. In the second type of column, the relatively high-viscositygel is “fixed” inside the capillary, usually chemically anchored to the capillary wall surface. The first column type is frequently applied to double-stranded (ds) DNA (e.g., PCR product) analysis; the latter capillary column type is typically used for analyzing singlestranded (ss) DNA (primers and probes), including antisense DNA. With replaceable gels, sample introduction can be made by either hydrodynamic (pressure, vacuum) or electrokinetic means. The hydrodynamic injection is generally preferred in quantitative work. Here, the composition of the sample introduced as a plug into the capillary should be exactly that of the original sample. With the electrokinetic injection, mobility differences between sample components cause a sampling bias; high-mobility Beckman Instruments, Inc. Palomar Analytical Services. (1) Guttman, A In Handbook of Capillay Electrophoresis; Landers, J. P., Ed.; CRC Press: Boca Raton, FL, 1994; p 129. (2) Pariat, Y. F.; Berka, J.; Heiger, D. N.; Schmitt, T.; Cohen, A S.; Foret, F.; Karger, B. L. J. Chromatogr. A 1993, 652, 57. +
0003-2700/95/0367-2279$9.00/0 0 1995 American Chemical Society
analytes are preferentially introduced into the capillary over slowmoving ones.3~~ In addition, effects due to the presence of salt or other substances in the sample must be carefully considered. For example, when electrokinetic injections are made from samples containing different salt concentrations, the amount of analyte detected may vary considerably.5 This may directly affect quantitation, e.g. in the accuracy of a drug assay: necessitating the use of external and/or internal However, with the use of nonreplaceable, fixed gels, electrokinetic sample introduction is the only injection mode feasible, as the high-viscositygel prevents aqueous sample plugs from entering the capillary. Clearly, many earlier problems affecting precision and accuracy (e.g., those related to voltage, temperature and electroosmoticflow control) with “manual”capillary electrophoresis (CE) instruments have been overcome by the emergence of automated sample introduction systems4t9and commercial CE instrumentation.10 Butler et a1.8 recently compared the precision feasible with CGE with other methods (slab gel, hybridization, spectrophotometry) for the quantitation of PCR products. Using an internal standard, precision values of -0.1% and -3% relative standard deviation (RSD) were reported for migration times and peak areas, respectively. However, as is evident from the recent CE literature, several peculiar effects related to sample introduction and matrix effects are still reported. Grushka and McCormickll calculated a “maximum allowable plug length” based on the solute’sdiffusion coefficient and analysis time. The authors found that the insertion of the capillary into a sample vial may result in an inadvertent injection. A detailed study of this effect, caused by an interfacial pressure difference at the inlet of the capillary, was recently (3) Huang, X.; Gordon, M. J.; Zare, R N. Anal. Chem. 1988, 60, 375. (4) Rose, D. J., Jr.; Jorgenson, J. W. Anal. Chem. 1988, 60, 642. (5) Satow, T.; Machida, A; Funakushi, IC; Palmieri, R J. High Resolut. Chromatogr. 1991, 14, 276. (6) Srivatsa, G. S.; Batt, M.; Schuette, J.; Carlson, R H.; Fitchett, J.; Lee, C.; Cole, D. L. J. Chromatogr. A 1994, 680, 469. (7) Dose, E. V.; Guiochon, G. A Anal. Chem. 1991, 63, 1154. (8) Butler, J. M.; McCord, B. R; Jung, J. M.; Wilson, M. R; Budowle, B.; Allen, R 0 .J. Chromatogr. B 1994, 658, 271. (9) Schwartz, H. E.; Melera, M.; Brownlee, R. G. /. Chromatogr. 1989, 480, 129. (10) Warner, M. Anal. Chem. 1994, 66, 1137A (11) Gmshka, E.; McCormick, R M. J. Chromatogr. 1989, 471, 421.
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published by Fishman et al.12J3Peak broadening due to penetration of residual analyte from the outside of the capillary was described by Lux et al.I4 By simply dipping the capillary in buffer after sample introduction, the problem can be prevented. Ermakov et al.15 observed artifactual peak splitting due to interaction of sample and background electrolyte. In the analysis of dsDNA with pressure injection, sample matrix effects were investigated by van der Schans et a1.16 The injection of an additional plug of a low-resistance solution (e.g., 0.1 M Tris-acetate) prior to the sample resulted in improved resolution. During the course of our work with CGE, we have experienced two injection-related problems which may affect precision and accuracy as well as separation performance. The first problem (chemical artifacts) manifests itself with repetitive electrokinetic injections from the same sample vial. With successive injections, progressively smaller amounts of sample are introduced into the capillary, resulting in decreasing peak heights and areas. This injection problem was especially pronounced when the sample introduction was performed from sample volumes of -10-200 pL. This phenomenon was briefly mentioned in a paper dealing with PCR product analysis on a replaceable gel matrix17and was experienced also with fixed gel capillaries.Is With capillary zone electrophoresis (CZE), Rose and Jorgenson4 briefly mentioned the possibility of sample contamination by the formation of electrochemical reaction products result&g from the flow of current through the sample solution. However, no thorough explanation of or solution to the problem has been reported so far. The second injection problem in CGE bhysical artifacts) we report involves the physical shape of the capillary end. A nonperpendicular edge (e.g., due to a poor cut during the manufacturing process) at the inlet side of the capillary results in substantial peak distortion, causing serious loss in resolution, whereas a capillary with a straight edge does not. In this technical note, we investigate these effects and propose simple ways to prevent these problems. EXPERIMENTAL SECTION
In all studies, a P/ACE system 2100 CE apparatus (Beckman Instruments, Inc., Fullerton, CA) was used in reversed polarity mode (cathode on the injection side). The separations were monitored on-column at 254 and 230 nm for the electrokinetic injection (oligonucleotide) and the column edge (naproxen) experiments, respectively. The temperature of the cartridge containing the polymer network-filled capillary column was thermostated at 20 f 0.1 “C by the liquid cooling system of the P/ACE instrument. The electropherograms were acquired and stored on an IBM 486/66 MHz computer and were evaluated with System Gold software (Beckman Instruments, Inc.). (12) Fishman, H. A;Amudi, N. M.; Lee, T. T.;Scheller, R H.; a r e . R N. Anal. Chem. 1994,66, 2318. (13) Fishman, H. A;Scheller, R. H.; Zare, R. N. J Chromatogr. A 1994,680, 99. (14) Lux, J. A;Yin, H. F.; Schomburg, G. Chromatographia 1990,30, 7. (15) Ermakov. S.V.; Zhukov, M. Y.; Capelli, L;Righetti, P. G.Anal. Chem. 1994, 66,4034. (16) van der Schans, M. J.; Allen, J. K; Wanders, B. J.; Guttman, A J Chromatoq. A 1994,680, 511. (17) Schwartz, H.E.;Lrlfelder, K; Sunzeri, F. J.; Busch, M. P.; Brownlee, R G. J. Chromatogr. 1991,559, 267. (18) Guttman, A; Ohms, J. I.; Cooke, N. High resolution separations of oligonucleotides using capillary gel electrophoresis; Third Intemational Symposium on High Performance Capillary Electrophoresis, San Diego, CA. 1991; Poster PT-11.
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Number of Injections Figure 1. Peak height of the p(dA)SO (absorbance units) versus numbers of consecutive injections. Inset: Capillary gel electrophoresis separation of the test mixture of p(dA)40-60; time frame, 20-27 min. Conditions: eCAP-ssDNA-100 gel-filled capillary column; E = 300 V/cm; t = 20 “C; sample, 7.4pglmL total DNA in water; injection, (W) 1.5 s, 7.5 kV electrokinetic injection from sample vial (“regular injection”), and (0)1.5 s, 7.5 kV electrokinetic injection from sample vial preceded by a 1.5 s, 7.5kV electrokinetic injection from water (“water preinjection”).
In the oligonucleotide separation experiments the eCAF’ssDNA-100 kit (Beckman Instruments, Inc.) was used with a 100 pm id., 30 cm effective length (37 cm total length) coated capillary tubing filled with a high-concentration, non-cross-linked, linear polyacrylamide gel in 100 mM Tris-borate (PH 8.5) buffer. The use of a coated capillary in conjunction with the polymer network results in negligible electroosmotic flow (EOF). A 0.2 OD (7.4 pg/mL total DNA with Na as counterion) solution of poly(deoxyadenylic acid), p(dA)~o-~), supplied with the kit was dissolved in deionized water to the final concentration of 0.5 pM. During the electrophoresis, an electric field of 300 V/cm was applied to the gel-filled capillary columns in all the experiments. In order to remove impurities from the polyacrylamide gel, the capillary column was pre-electrophoresed with the appropriate running buffer at 100,200, and 300 V/cm for 10 min each. The samples were injected electrokinetically onto the column by applying a power of 0.124 W (voltage, 7.5 kV; current, 11 p& injection time, 1.5 s). The tip of the capillary was immersed into deionized water before and after the injections to avoid any buffer and sample carryovers. The pH change of the DNA samples during the different injection procedures was measured with a Beckman +72 pH meter equipped with a microelectrode. For the capillary edge evaluation experiments, 25 pm id., 375 pm o.d., 20 cm effective length (27 cm total length) untreated fused silica capillary tubing was used. The 25 pm id., 375 pm 0.d. capillary was selected to investigate the worst possible case (i.e., where the free silica surface to volume ratio is the largest). Ultrapure grade 200 mM 2-(N-morpholino)ethanesulfonic acid (MES) was used for the buffer, adjusted to pH 5.0 with tetrabutylammonium hydroxide (ICN, Costa Mesa, CA). The R,S-naproxen was used in a 1O:l enantiomeric ratio during the experiments and was a kind gift of Prof. Gyula vigh, Texas A&M University, College Station, TX. The poly(ethy1ene oxide) (MW 300000) was purchased form Sigma (St. Louis, MO) and the hydroxypropyl-b-cyclodextrin(HP;O-CD) was from American Maize (“on, AZ). The gel-buffer system contained 200 mM
DNA Zone Injected
Figure 2. Schematic diagrams illustrating the sample introduction processes. (A) Regular electrokinetic injection. DNA enters the capillary. Hydroxyl ions are generated at the electrode. Tris ions migrate from the capillary into the sample vial. Borate migrates toward the anode. At the end of this injection, a DNA zone has been introduced into the capillary. (6) Electrokinetic injection from water (water preinjection). Tris (T-) and borate (B-) migrate to the cathode and anode, respectively. Hydroxyl ions are generated at the cathode. An ion-depleted zone (high resistance, low conductivity) is created at the end of the capillary. (C) Electrokinetic sample injection with prior preinjection of water. The ion-depleted zone at the end of the capillary results in a larger DNA zone to enter the capillary.
MES/TBAH, 10 mM HP-p-CD and 0.4% polymeric additive, pH 5.0. The use of a low-viscosity polymer network permitted replacement of the gel-buffer system in the capillary column by means of the pressure rinse operation mode of the P/ACE apparatus (i.e., replaceable gel). The tip of the oblique edge capillary was rasped to a 45” angle under a microscope using a cutting tool. The polyimide outside coating was removed from the end (5 mm) of the column with a razor blade to avoid any sample or buffer trapping between the fused silica capillary and the coating. The samples were injected by pressure (injection time, 3 s; pressure, 0.5 psi = 3447.4 Pa) into the replaceable gelfilled capillary column. Similar to the DNA experiments, the tip of the capillary was immersed into deionized water before and after the injection to avoid any carryovers. The samples were either stored at -20 “C or freshly used. All buffer solutions were filtered through a 0.45 pm pore size filter (Schleicher and Schuell, Keene, NH) and carefully vacuum degassed before use. RESULTS AND DISCUSSION
Chemical Artifacts. In most applications of CE, the injection volumes are small, typically -1-100 nL. Overloading the capillary, i.e., introducing a sample plug which is too large, results in extra zone broadening,” unless stacking conditions with discontinuous buffers prevail.1g The small volume required for introduction into the capillary can be especially advantageous when sample is available only in limited quantities (low microliter volumes). In this case, however, it is difficult to aliquot the original sample. Therefore, multiple injections (needed to increase the confidence of the analytical results and precision studies) have to be performed from the same sample vial. The inset in Figure 1shows the separation of a p(dA)40-~test mixture on a high-viscositygel-filled capillary column. The sample was electrokinetically (7.5 kV, 1.5 s) injected from a 50 pL, 0.2 OD (-0.5 pM) solution placed in a 400 pL polypropylene minivial (“regular injection”). Consecutive injections from this vial resulted (19) Chien, R-L; Burgi, D.S.Anal. Chen. 1992, 64,489A.
in an observed decrease in peak absorbance and peak area, Le., a decrease in the amount of sample introduced into the capillary. In Figure 1 (lower trace), this is expressed as an exponential decrease in absorbance of the maximum peak, p(dA)so, of the polydeoxyadenylic acid sample over five consecutive runs. Note that injection number 5 had a 70% lower peak height than that of injection number 1. The decrease in absorbance was less pronounced when the sample volume was increased. The same experiment using an 80x larger sample volume (4 mL) did not result in a significant decrease in peak absorbance (results not shown). It should be noted that the same decreasing trend in sample amount was also observed with other types of CGE columns and DNA samples, e.g., with consecutive electrokinetic injections of dsDNA on replaceable polymer network c ~ l u m n s . ~ ~ J ~ For both the replaceable and the fixed gel columns, similar experimental CE conditions apply, such as similar Tris-borate buffer, coated capillary, no EOF, and reversed polarity. Figure 2A sketches the situation of the first sample injection. Upon applying voltage, negatively charged DNA is drawn into the capillary. OH- is generated at the cathode according to the electrochemicalreaction 2H20 2e 20H- HZand is moving into the capillary toward the anode. Tris migrates out of the capillary and into the sample solution. During the injection process, incoming hydoxyl ions will shift the TH+ == T H+ equilibrium to the right, thereby reducing the effective mobility of Tris. Borate migrates toward the anode. The amount of hydroxyl ions generated in the sample vial during the electrolysis can be estimated from Faraday’s law and would equal 3.4 pM, corresponding to pH 8.5. With consecutive injections, the hydroxyl and Tris concentrations as well as the pH in the sample vial will continue to increase (electrical resistance decreases). Using a micro-pH meter, the pH in the sample solution was measured. During the series of injections, the pH in the sample solution was found to increase from 7.1 to 8.5 (-20% increase). After the hydroxyl concentration reaches an appreciable number, DNA (sample concentration,0.5 pM) will compete with hydroxyl for introduction into the capillary. As hydroxyl is more mobile
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m2V-’ s-l; p o = ~ 200 x m2V-’ than DNA @DNA = 5 x s-9, it serves as a more efficient current carrier than the DNA. Consequently, the amount of DNA injected with consecutive injections will decrease, as indeed is seen in Figure 1. A simple solution to the above problem is to perform an electrokinetic injection from water just prior to the actual sample injection (Figure 2B; the capillary was positioned between two vials containing water; the injection time was the same as that of the sample injection). During this “preinjection”,Tris migrates into the water vial, while OH- is generated at the cathode. An “ion-depleted zone (i.e., a relatively high-resistance,lowconductivity zone) is created at the end of the capillary. Subsequently, the capillary-with its ion-depleted zone-is positioned into the sample vial for the second (DNA) injection (Figure 2C). pH measurements revealed only a 2% increase in the pH of the sample vial after five injections carried out in this fashion (as opposed to a 20%increase without the preinjection of water, see above). An important consequence of the introduction of a relatively high-resistance,lowconductivity zone into the capillary is that the local field strength in this zone is relatively high. Hence, more sample is pulled into the capillary than would occur without the intermediate water injection.19 With the intermediate water injection technique, a substantial increase in W absorbance (peak height) is observed (2-fold for the first and a 5fold for the last injection; compare traces in Figure 1). A similar double injection was performed by Ulfelder et aLZoand Butler et al.* with a replaceable gel matrix (alkylcellulose solutions). In these experiments, the water plug was introduced by hydrodynamic means (pressure), with the object to maximize sample detectability for low-copy DNA applications. The slightly decreasing trend in the upper trace of Figure 1 suggests that the water injection did not completely eliminate the (exponential) decrease in absorbance shown in the lower trace. Perhaps small amounts of Tris-as well as hydroxyl generated at the cathode-still migrate into the sample vial, affecting the sample load with each subsequent injection. However, precision is greatly improved compared to the experiments without the intermediate water injection. The data points of Figure 1 (upper trace) resulted in a RSD peak height of 4.0% (five consecutive injections), typical of the precision achievable with CGE without internal standards.6 Optimization of the preinjection and sample injection times may further increase precision. In addition, the presence of salt in the sample (common with PCR samples) will affect sample l ~ a d a b i l i t y . Work ~ ~ ~ ~is~ ~in~ progress ~ in our laboratory to investigate these effects. Encouraging preliminary results have been obtained with the abovedescribed electrokinetic injection technique for nondesalted PCR samples, resulting in excellent DNA peak Physical Artifacts. In recent years, various “physical”,injection-related effects, affecting separation performance, have been discussed in the literature. With micellar electrokinetic capillary chromatography (MECC), Lux et all4 found that during the injection process, “carryover”liquid at the end of the capillary results in peak asymmetry; the authors suggested an additional rinsing step in the sample introduction procedure. With CZE, Fishman et al.12J3studied an “ubiquitous”injection occurring when a capillary just touches a sample solution. The interfacial pressure difference across the curved surface of the droplet at the end of (20) Welder, K. J.; Schwartz, H.
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Figure 3. Effect of the actual physical shape of the capillary inlet on the resolution of naproxen enantiomers. Test solute: 0.1 mg/mL (Rj-naproxen and 0.01 mg/mL (S)-naproxen in water. (A) Properly cut (perpendicular to the axis of the capillary) column inlet and (e) oblique (45”) cut column inlet.
the capillary is causing a small amount of sample to be pulled into the capillary. In work with CGE, employing noncross-linked, low-viscosity polymer networks, we found impaired separation performance with capillaries which were improperly cut at the inlet side. Specifically,an oblique inlet side of the capillary yields a separation efficiency (expressed as the theoretical plate number, N) significantly lower than that obtainable with a properly cut, straight edge capillary, causing loss in resolution with similar separation selectivities. Recently, a similar phenomenon was independently confirmed by Grushka and cc-workers23 in their work with CZE. Hence, it appears that the sample plug must be positioned accurately at the inlet in such a way that its boundaries are perpendicular to the axis of the column during the injection process. Assuming otherwiseidentical injection conditions, in the case of oblique edge capillaries, the injection plug length is slightly increased (relative to the straight edge capillary), resulting in increased extracolumn variance due to the injection plug.23 Figure 3 shows the chiral separation of naproxen enantiomers, using pressure injection onto a properly (A) and an improperly (B) cut, replaceable gel-filled capillary. Naproxen, one of the most frequently prescribed chiral drugs, was used as a test solute in
E.;Hall, J. M.;Sunzeri, F. J. Anal. Biochem.
1992,200,260.
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(21) Hjerten, S.; Valtcheva, L.;Li, Y.-M. J Cap.Elecfrophor. 1994, I , 83.
an enantiomeric ratio of WS 101. 'The chiral selector HP-p-CD was used to attain enantiomeric separation in a low-mobility buffer system (MESITBAH). In the case of Figure 3B, the inlet-side edge of the capillary was cut with an -45" angle, as shown in the inset. Note that, under otherwise equivalent injection and separation conditions, with the use of an oblique edge capillary, severe peak distortion occurs, resulting in >60%loss in peak efficiency for both of the enantiomers (41 200 vs 15 500 theoretical plates for the R, and 73 500 vs 17 700 theoretical plates for the S enantiomers). Likewise, enantiomeric resolution decreased from R, = 1.57 to 0.97. Thus, careful column edge cutting plays an important role in achieving optimum (Gaussian) peak shape in CGE. In practice, this may not be that important when dealing with well-separated peaks (Rs> 3) of similar height. However, the effect may be substantial in trace analysis, e.g., when determining trace impurities in protein preparations with SDSbased replaceable gel columns, for closely related PCR fragments, or especially for chiral compounds with the recent FDA requirement of the analysis of high enantiomeric excess ratios (looO:l).24In these instances, employing an oblique injection edge capillary, the trace analyte (22) Schwartz, H. E.; Welder, K J.; Guttman, A Injection Related Artifacts in Capillary Electrophoreseis; Seventh International Symposium on High Performance Capillary Electrophoresis, Wlirtzburg, Germany, 1995; Poster P428. (23) Cohen, N.; Gmshka, E. /. Chromatog. A 1994, 684, 323. (24) Guttman, A; Cooke, N. J Chromatogr.A 1994, 685, 155.
will not be sufficiently resolved from the main peak in order to perform accurate quantitation. CONCLUSIONS In summary, two injection-related artifacts in capillary gel electrophoresis were examined. 'The first occurs with consecutive electrokinetic injections from small volume aqueous samples and results in progressively smaller analyte peak heights and areas. The problem can simply be prevented by adding an intermediate water injection step in the CGE run sequence. The second problem relates to the capillary inlet shape of the gel-filled column. Here, careful cutting (perpendicular to the axis of the column) and examination of the capillary edge prevents any undesirable peak distortion and consequent resolution loss.
ACKNOWLEWMENT The support of this work by Dr. Nelson Cooke is highly appreciated. The authors gratefully acknowledge Professor Eli Grushka and Dr. Bart Wanders for their stimulating discussions. Received for review December 13, 1994. Accepted April 11, 1995.@ AC941206J @
Abstract published in Advance ACS Abstracts, June 1, 1995.
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